The present invention relates to the use of cytohesin-PH peptides to influence the ability of integrins to adhere.
T-Lymphocyte activation is achieved by coordinated binding of adhesion molecule receptors and signal receptors which are then expressed on the surface of T cells when these receptors bind to their complementary receptors on the antigen-presenting cell. Besides the T-cell receptors (TCRs) and MHC (major histocompatibility class) class I or II proteins, which are always involved in leukocyte activation, various types of coreceptors also are necessary, such as the integrins, and the CD2, CD4 and CD8 molecules. The functional interaction between the TCRs and the T-lymphocyte coreceptors is dynamic in nature, that is, only the binding of a TCR to its target molecule brings about enhanced binding of the coreceptors to their complementary receptors.
The integrins are a large family of cell surface molecules. These molecules are heterodimers that comprise pairs of xcex1 and xcex2 chains without disulfide linkages. Because there are several different xcex1 and xcex2 chains, differences in ligand specificity are achieved by different combinations of the xcex1 and xcex2 chains. The integrins are involved both in direct cell-cell interaction and in the binding of cells to the extracellular matrix.
Integrins that occur on non-activated lymphocytes are in a so-called xe2x80x9clow-avidity state,xe2x80x9d which is converted very rapidly by T-cell activation into a transient so-called xe2x80x9chigh-avidity state.xe2x80x9d The mechanism of this so-called xe2x80x9cinside-out signalingxe2x80x9d has not yet been elucidated, however. Collins et al. Curr. Opinion Imm. 6: 385-393 (1994).
According to the affinity modulation model on T-cell activation, there is a conformational change in the integrins which first makes the high-affinity ligand binding site accessible to the ligand. Possible molecular events bringing about the conformational change which are currently suggested are covalent modification (for example, phosphorylation) or binding of activator or repressor molecules to the cytoplasmic domain of the integrin xcex2 subunit, but there is no experimental evidence in favor of a particular mechanism. Diamond and Springer, Curr. Biol. 4: 506-517 (1994).
Another type of signal protein is the hsec7hom (human SEC7 homolog) protein, which is mainly expressed in natural killer cells and cytotoxic T cells. This protein was thought to be the human homolog of the SEC7 protein from S. cerevisiae. However, because of the (i) great difference in the molecular weights of SEC7 and hsec7hom, (ii) the sequence similarity that is limited to a relatively short section, and (iii) the specific expression of hsec7hom, it is now thought that hsec7hom does not belong to the SEC7 protein family. See Liu and Pohaidak, Biochimica et Biophysica Acta 1132: 75-78 (1992)) For these reason, the hsec7hom protein will be referred to as xe2x80x9ccytohesin-1.xe2x80x9d
Cytohesin-1 contains two regions which are homologous with domains of other proteins:
1. SEC7 domain: this domain contains about 200 amino acids and is only known to be found in a few other proteins. One of these proteins is the SEC7 protein, which is involved in secretion in yeasts. Another protein that possesses this domain is EMB30, which is involved in embryogenesis in Arabidopsis. Shevell et al., Cell 77: 1051-1062 (1994).
2. PH domain (Pleckstrin homology domain): this domain is about 100 (xc2x125) amino acids long and has been found in a number of proteins, many of which play a part in the signal transduction. The three-dimensional structure of some PH domains has been elucidated. These domains are able to function as ligand-binding domains. Tsukada et al., Proc. Nat""l Acad. Sci USA 91: 11256-60 (1994). Although it has been shown that the heterotrimeric G proteins can interact with PH domains, no exact physiological function for PH domains has been previously found. Birney, TIBS 19: 349-353 (1994). Because the C-terminus of the PH domain has not been conclusively determined, larger amino acid sequences can be employed to ensure that the entire PH domain is present.
The PH domain is of major importance with regard to the present invention because of its ability to interact with the integrins.
The integrins are found on leukocyte surfaces and are involved in the inflammation process. Within minutes after receiving an inflammatory stimulus, the integrins acquire, through signal transduction pathway(s), the ability to attach to cell-surface and extracellular ligands. In some cases, the activation is transient, which means that the integrins quickly lose the ability to adhere. The dynamic cycling between adhesive and non-adhesive states endows a cell with the ability to rapidly regulate adhesion to ligands on apposing cell surfaces and matrices. This ability may be implicated in cell movement, which requires a rapid flux of adhesive interactions.
The function of integrin adhesion was initially documented in experiments that interfered with integrin function by using antibodies of peptide antagonists. The physiology of integrins has been assessed by the investigation of natural or induced genetic mutations of individual subunits. These mutations result in a variety of pathological sequelae.
Integrin-mediated adhesions has functional roles in a wide variety of biological and pathological settings, including hemostasis, inflammation, and tumor metastasis and development. For example, in primary hemostasis, platelet attachment to blood vessel walls, and aggregation at the site of injury are mediated by the integrins. Adhesion and signal transduction by integrins are essential elements of a sequence of intracellular interactions leading to antigen-specific activation of T-lymphocytes.
In inflammation, integrins mediate the critical attachment-strengthening step in the adhesion cascade, which permits leukocytes to move from the vasculature, across the endothelium lining blood vessels, and into the parenchyma. The subsequent migration of cells through the parenchyma depends upon the transient nature of integrin adhesiveness. This migration also may depend upon a sequence of attachment and detachment of ligand(s) by rapidly activated and inactivated integrin subpopulations, which are located and the leading and trailing edges of the migrating cells.
Because the vast array of functions performed by the integrins, these molecules are implicated in a large number of disease states. Accordingly, there is a need for methods of influencing the ability of integrins to adhere. This need is satisfied by the present invention.
It is an object of the present invention to provide methods of influencing the ability of integrins to adhere.
It is another object of the present invention to provide methods of influencing the ability of integrins to adhere by employing cytohesin-PH.
It is still another object of the present invention to administer proteins that contain the cytohesin-PH peptide to patients to treat diseases and otherwise improve the physical condition.
It is yet another object of the present invention to provide assays, including those to screen drugs, using proteins that contain the cytohesin-PH peptide.
In accomplishing these and other objects, there is provided, in accordance with one aspect of the invention, the use of a cytohesin-PH peptide, in particular as shown in FIG. 2 (SEQ ID NO: 12) or parts of the sequence shown therein, such as amino-acid positions 258 to 398, (residues 258 to 398 of SEQ ID NO: 12) to regulate the T-lymphocyte activation.
The invention furthermore relates to the use of a DNA coding for a cytohesin-PH peptide, in particular as shown in FIG. 2 (SEQ ID NO: 11) or parts of the sequence shown therein, such as nucleotide positions 841 to 1263, (bases 841 to 1263 of SEQ ID NO:11) for expression of the peptide.
The invention furthermore relates to the use of a DNA whose sequence is degenerate (often referred to as codon/anticodon wobble) with respect to the sequence of the DNA mentioned above in accordance with the nature of the genetic code.
The invention furthermore relates to the use of a DNA which hybridizes under stringent conditions with the DNA shown in FIG. 2 (SEQ ID NO: 11). Such DNAs include probes, which can be used to identify and/or isolate a gene or other nucleotide sequence. One type of DNA according to the invention would hybridize to the DNA of FIG. 2 (SEQ ID NO: 11) under highly stringent conditions.
The invention furthermore relates to vectors comprising a DNA described above and the use thereof for the expression of a cytohesin-PH peptide.
The invention furthermore relates to host cells comprising one of the vectors described above, and uses thereof.
The invention additionally relates to the use of a cytohesin-PH peptide described above for reducing or otherwise influencing inflammations, for improving wound healing, for suppressing the immune system, in particular in organ transplants, for preventing metastasis of hematopoietic tumors and for treating arteriosclerosis.
The invention furthermore relates to a pharmaceutical comprising a cytohesin-PH peptide and a physiologically acceptable vehicle and, where appropriate, suitable additives and/or ancillary substances.
The invention furthermore relates to an assay system with relevance for therapeutic use comprising the cytohesin-PH domain, preferably a drug screening assay system.
The invention furthermore relates to a cytohesin-2 peptide having the amino-acid sequence shown in FIG. 1B (SEQ ID NO: 10). The invention additionally relates to the use of a cytohesin-2 peptide having the amino-acid sequence encoded by the cts 18.1-cDNA (SEQ ID NO: 14) to regulate T-lymphocyte activation. The invention additionally relates to a DNA coding for a cytohesin-2 peptide or parts thereof. A sample of the cts 18.1 cDNA has been deposited at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany. The deposit has been assigned accession number DSM 13656.
The invention furthermore relates to a DNA whose sequence is degenerate with respect to the sequence of the DNA mentioned above in accordance with the nature of the genetic code. Degeneracy is often referred to as codon/anticodon wobble, and is discussed in Watson et al., MOLECULAR BIOLOGY OF THE GENE (4th ed. 1987) at 437-43. Also within the scope of the invention are so-called xe2x80x9cpolyamidexe2x80x9d or xe2x80x9cpeptidexe2x80x9d nucleic acids (xe2x80x9cPNAsxe2x80x9d), which replace the (deoxy) ribose phosphate backbone with an achiral polyamide backbone or the like. See Nielsen et al., Science 254: 1497-54 (1991).
The invention furthermore relates to the use of a DNA coding for a cytohesin-2 peptide for expression thereof. The invention furthermore relates to the use of a cytohesin-2 peptide as shown in FIG. 1B (SEQ ID NO: 10) for influencing inflammations, for improving wound healing, for suppressing the immune system (in particular in organ transplants), for preventing metastasis of hematopoietic tumors and for treating arteriosclerosis.
The invention furthermore relates to a pharmaceutical comprising a cytohesin-2 peptide and a physiologically acceptable vehicle and, where appropriate, suitable additives and ancillary substances.
The invention furthermore relates to a process for the preparation of a cytohesin-PH peptide described above, which comprises:
(a) cultivating a host cell containing DNA encoding a cytohesin-PH peptide, and
(b) isolating the cytohesin-PH peptide.
Another aspect of the invention includes methods of regulating T-lymphocyte adhesion in a patient, comprising the step of administering to the patient an amount of a cytohesin-PH peptide. The cytohesin-PH peptide has an amino-acid sequence as shown in FIG. 2 (SEQ ID NO: 12), such as shown at positions 258 to 398 of FIG. 2 (residues 258 to 398 of SEQ ID NO:12). The method can be used to treat inflammation, improve wound healing, regulate the immune system (including suppression for organ transplant patients), treat hematopoietic tumors, and/or treat arteriosclerosis.
In accordance with still another aspect of the invention, there are provided methods of making a cytohesin-PH peptide for regulating T-lymphocyte adhesion, comprising the step of expressing a polynucleotide that hybridizes under stringent conditions with the DNA shown in FIG. 2 (SEQ ID NO: 11). The method can employ the sequence set forth at FIG. 2 (SEQ ID NO: 11), or portions thereof, such as the sequence set forth at positions 841 to 1263 of FIG. 2 (bases 841 to 1263 of SEQ ID NO: 12).
In accordance with yet another aspect of the invention, there are provided a cytohesin-2 peptides having the amino-acid sequence shown in FIG. 1B (SEQ ID NO: 10).
In accordance with yet a further aspect of the invention, there are provided pharmaceutical compositions comprising a cytohesin-PH peptide and/or a cytohesin-2 peptide along with a physiologically acceptable carrier. The pharmaceutical preparations can further comprise suitable additives and ancillary substances. Additionally, the pharmaceutical composition can be composed of cytohesin-PH and/or cytohesin-2 peptides as the only ingredient(s) that affect integrin adhesion.
In accordance with still a further aspect of the present invention, there are provided polynucleotides encoding a cytohesin-PH peptide and/or a cytohesin-2 peptide. The polynucleotides can hybridize under stringent conditions with the DNA shown in FIG. 2 (SEQ ID NO:11). The polynucleotides encoding the cytohesin-PH peptide can comprise the sequence set forth at FIG. 2 (SEQ ID NO: 11), or portions thereof, such as positions 841 to 1263 of FIG. 2 (bases 841 to 1263 of SEQ ID NO:11).
In accordance with still a further aspect of the present invention, there are provided assay kits comprising a cytohesin-PH peptide or as cytohesin-PH peptide. The assays kits can be used for drug screening, among other things.
In accordance with yet a further aspect of the present invention, there are provided methods of evaluating the effects of compounds, comprising the steps of contacting a compound with a cytohesin-PH peptide and determining the effects of the compound on the activity of cytohesin-PH. One type of assay would include cytohesin-PH or the test compound, wherein only one is bound to an insoluble matrix, such as SEPHAROSE. The other is labelled (radioactively, enzymatically, magnetically, or other appropriate labels), and a direct binding assay is conducted. This assay is capable of identifying compounds that bind to, and thus possibly block or inhibit, cytohesin-PH. Compounds that bind cytohesin-PH should then be tested with the cellular assays, described below.
Methods for evaluating the effects of compounds with cytohesin-PH also are provided. Such methods include cellular assays. A cellular assay could comprise the steps of: growing a test group and a control group cells that possess the ability to adhere to a substrate (such as a culture dish coated with ICAM-1-Rg or the like), wherein the test group is grown in the presence of a test compound and the control group is grown in the absence of a test compound; inducing the expression of cytohesin-PH in the test cells and the control cells; and comparing the extent of adhesion loss by the test group and the control group. In a valid test, the control group cells would lose adhesive capabilities. If the cells of the test group lose adhesive capabilities to a lower degree, the test compound interferes or blocks the anti-adhesive properties of cytohesin-PH.
The invention also includes the experimental steps which are explained by way of example are listed hereinafter:
1) Preparation of the CD18 cyt bait construct;
2) Preparation of the yeast expression bank;
3) Screening with the two-hybrid system;
4) Test of the binding specificity in yeast;
5) Preparation of the fusion constructs for testing the function of cytohesin-1 in vivo;
6) Function assay for cytohesin-1 and the subdomains
7) Preparation of the ICAM-Rg fusion protein; and
8) Cytohesin-PH domain-specific functional inhibition of xcex22 integrins.
The present invention encompasses biotechnology inventions, including biotechnological processes.
Still other aspects of the invention will be apparent to the skilled person in view of the teachings contained herein.